Granted Patent B2

US 7,375,188 · App. 11/194,093 · Granted May 20, 2008

Abstract

Claims (37)

1. A method for producing vegetarian Protein A comprising:

fermenting a secretor strain of Staphylococcus aureus ( S. aureus ) in a media containing yeast extract, vegetarian amino acids and peptides, glucose, and essential salts, said media being free of animal product, to produce a Protein A-containing media;

harvesting the Protein A-containing media; and

purifying the vegetarian Protein A by applying the Protein A-containing media to a synthetic anion exchange chromatography resin lacking animal products and eluting the Protein A from the resin followed by contacting with a ceramic hydroxyapatite chromatography column and eluting the Protein A from the column.

2. The method of claim 1 wherein the S. aureus is the secretor strain Staph Imre.

3. The method of claim 2 wherein the S. aureus are fermented for 10 to 24 hours.

4. The method of claim 3 wherein the S. aureus are fermented for 13 to 14 hours.

5. The method of claim 4 wherein the pH of the Protein A-containing media is adjusted to about 7.5 to 8.5 and the conductivity adjusted to less than 2 milliSiemens/centimeter by diafiltration using 10 millimolar TrisHCl and NaCl.

6. The method of claim 5 wherein the anion exchange chromatography is performed at about a 5.7 to 7.9 centimeter/minute flow rate on a column about 24 centimeters high.

7. The method of claim 6 wherein the Protein A is eluted from the chromatography column using at least 20 millimolar TrisHCl and NaCl at a conductivity of about 8 milliSiemens/centimeter.

8. The method of claim 7 wherein the pH of the Protein A elutant is adjusted to about pH 6.5 to 7.1 and a conductivity of about 1 to 2 milli-Siemens/centimeter by diafiltration with 14 millimolar sodium phosphate at pH 6.8.

9. The method of claim 8 wherein the Protein A is diafiltered using a membrane having 10,000 molecular weight cut-off point.

10. The method of claim 9 further comprising concentrating the Protein A filtrate to about 5 to 20 milligrams/milliliter using a 10,000 molecular weight filter and adjusting the Protein A solution to a neutral pH.

11. The method of claim 8 wherein the pH of the Protein A solution is adjusted to neutral pH using a phosphate buffer.

12. The method of claim 9 wherein the Protein A aplied to the hydroxyapatite chromatography column is washed with 14 millimolar sodium phosphate at pH 6.8 and eluted with about 370 mM sodium phosphate at pH 6.8.

13. The method of claim 12 wherein the height of the resin bed in the hydroxyapatite chromatoQraphy column is about 20 centimeters.

14. The method of claim 13 further comprising concentrating the Protein A elutant from the hydroxyapatite chromatography column to about 5 to 20 milligrams/milliliter using a 10,000 molecular weight filter and adjusting the Protein A solution to a neutral pH.

15. The method of claim 14 wherein the pH of the Protein A solution is adjusted to neutral pH using a phosphate buffer.

16. A method for producing vegetarian Protein A comprising:

fermenting Staph Imre in a media consisting essentially of yeast extract, soy peptides, glucose and essential salts, to produce a Protein A-containing media;

harvesting the Protein A-containing media;

purifying the Protein A by applying the Protein A-containing media to an anion exchange resin lacking animal products and eluting the Protein A from the resin.

17. The method of claim 16 wherein a Staph Imre seed used to inoculate a fermenter is maintained in vegetarian media.

18. The method of claim 17 wherein the Staph Imre are fermented for 10 to 24 hours.

19. The method of claim 16 wherein the Staph Imre are fermented for 13 to 14 hours.

20. The method of claim 19 wherein the pH of the Protein A-containing media is adjusted to about 7.5 to 8.5 and the conductivity adjusted to about less than 2.0 milliSiemens/centimeter using 10 millimolar TrisHCl and NaCl.

21. The method of claim 20 wherein the anion exchange chromatography is performed at about a 5.7 to 7.9 centimeter/minute flow rate on a column about 24 centimeters high.

22. The method of claim 21 wherein the Protein A is eluted from the chromatography column using at least 20 millimolar TrisHCl and NaCl at a conductivity of about 18 milliSiemens/centimeter.

23. The method of claim 22 wherein the pH of the Protein A elutant is adjusted to about pH 6.5 to 7.1 and conductivity of about 1.0 to 2.0 milliSiemens/centimeter by diafiltration with 14 millimolar sodium phosphate at pH 6.8.

24. The method of claim 23 wherein the Protein A elutant is diafiltered using a membrane having a 10,000 molecular weight cut-off point.

25. The method of claim 24 further comprising concentrating the Protein A filtrate to about 5 to 20 milligrams/milliliter using a 10,000 molecular weight filter and adjusting the Protein A solution to a neutral pH.

26. The method of claim 25 wherein the pH of the Protein A solution is adjusted to neutral pH using a phosphate buffer.

27. The method of claim 24 further comprising applying the Protein A filtrate to a hydroxyapatite chromatography column to remove non-protein contaminants and eluting the Protein A from the hydroxyapatite chromatography column.

28. The method of claim 27 wherein the Protein A filtrate applied to the hydroxyapatite chromatography column is washed with 14 millimolar sodium phosphate at pH 6.8 and eluted with about 370 mM sodium phosphate at pH 6.8.

29. The method of claim 28 wherein the height of the resin bed in the hydroxyapatite chromatography column is about 20 centimeters.

30. The method of claim 28 further comprising concentrating the Protein A eluant from the hydroxyapatite chromatography column to about 5 to 20 milligrams/milliliter using a 10,000 molecular weight cut-off filter and adjusting the Protein A solution to a neutral pH.

31. The method of claim 30 wherein the pH of the Protein A solution is adjusted to neutral pH using a phosphate buffer.

Assignments (21)